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Home , Agathisanthemum, Amaracarpus, Anthospermeae, Anthospermum, Arcytophyllum, Asperula

Plant Syst. Evol. 225:43-72 (2000) Systematics and Evolution © Springer-Verlag 2000 Printed in Austria

Phylogeny and classification of the subfamily ()

B. Bremer 1 and J.-F. Manen 2

1Department of Systematic Botany, EBC, Uppsala University, Uppsala, Sweden 2Universit6 de Gen6ve, Conservatoire et Jardin Botaniques, Chamb&y, Switzerland

Received April 27, 1999 Accepted June 21, 2000

Abstract. We performed phylogenetic analyses of Progress in understanding of the subfamily the subfamily Rubioideae (Rubiaceae) based on Rubioideae of the Rubiaceae is relatively three different pieces of chloroplast DNA, the recent and includes many important contribu- protein coding rbcL gene, the spacer sequence tions from many different scientists. Before the between atpB and rbcL (atpB-rbcL), and the recently middle of the 20th century the "Rubioideae" published (Andersson and Rova 1999) rpsl6 intron taxa were dispersed in the two subfamilies data. New rbcL sequences have been produced for Coffeoideae and Cinchonoideae, a classifica- 41 taxa and there are 52 new atpB-rbcL spacer sequences. All analyses gave similar results concern- tion of the Rubiaceae based on ovule number ing the phylogeny, but they differ slightly in reso- (Schumann 1891). Bremekamp (1952, 1954) lution and support for the various branches. The and Verdcourt (1958) argued against this minor tribes , Urophylleae, Lasian- artificial division of the and instead theae, and Coussareeae form a grade to the rest of proposed that all Rubiaceae tribes with the subfamily, which consists of two well-supported containing raphides (calcium oxalate crystals) branches, the alliance and the Sper- should be set aside as a new subfamily, macoceae alliance, including a majority of all genera Rubioideae. There are large similarities and species. Based on the resulting phylogenies we between the systems of these two authors. present a revised classification of the Rubioideae. Verdcourt included 16 tribes (and the unplaced We accept 16 tribes of which 12 more or less ) in the subfamily Rubioideae, and correspond to earlier tribal circumscriptions: , Argostemmateae, Craterisper- Bremekamp (1966), who used a more narrow meae, Gaertnereae, Morindeae, Paederieae, tribal circumscription, included 19 tribes and Psychotrieae, Schradereae, , , excluded some tribes to other subfamilies: Theligoneae, and Urophylleae; two tribes have Hillieae to Hillioideae, Ophiorrhizeae to received new and very different circumscriptions: Ophiorrhizoideae, and Urophylleae and Ophiorrhizeae and Coussareeae; and two are new to Pauridiantheae to Urophylloideae. Characters, science: Lasiantheae and Danaideae. except the raphides, that were used to distin- guish Rubioideae were valvate aestivation Key words: Rubiaceae, Rubioideae, Danaideae, (with few exceptions, Harnelia, , and Lasiantheae, chloroplast DNA, rbcL, atpB-rbcL Hillia - all these have later been shown to spacer, rpsl6 intron, phylogeny, classification. belong to Cinchonoideae; e.g. Bremer et al. 44 B. Bremer and J.-F. Manen: Rubioideae classification

1995) and indumentum of septated (articulat- (Bremer and Jansen 1991) and Hillieae (Bremer ed) hairs. The next major change in the et al. 1995, Natali et al. 1995) do not belong in Rubioideae circumscription was presented by the subfamily, that Theligoneae belong in the Robbrecht (1988). His Rubioideae with 16 Rubioideae close to Rubieae (Bremer 1996a, tribes agreed largely with Bremekamp's but Natali et al. 1996), and that belong Robbrecht included Ophiorrhizeae and Theli- in the Rubioideae and are not part of Anti- goneae (shown to be related to Rubiaceae by rheoideae (Bremer 1996a). Wunderlich 1971) and excluded Knoxieae Molecular investigation has proved to be and Craterispermeae to the subfamily very informative for reconstruction of phylo- Antirheoideae. These three classification geny but several tribes are yet to be represented schemes of Bremekamp, Verdcourt, and Rob- in molecular investigations. Just before brecht have been important sources of inspi- completion of this manuscript a detailed ration for later more focused studies of analysis of the Rubioideae based on the rpsl6 and morphology. Comprehensive intron was published (Andersson and Rova taxonomic studies of different Rubioideae 1999). That study confirms, in most parts, tribes that should be mentioned are those by earlier molecular studies (Bremer 1996a, Natali Puff and Robbrecht with co-authors (Puff et al. 1996) but as more taxa are included it 1982, 1988; Puff and Robbrecht 1989; Puff also contributes a lot of new information. and Buchner 1998; Robbrecht 1982; Robb- The present study aims at presenting new recht et al. 1991), and Johansson (1987a, b, phylogenetic analyses of the Rubioideae based 1988, 1989, 1994). Other examples are chro- on two different sequences of chloroplast mosome investigations by Kiehn (e.g. 1986, DNA, the protein coding rbcL gene and the 1995), morphological studies by Igersheim and spacer sequence between atpB and rbcL co-authors (Igersheim 1992, Igersheim and (atpB-rbcL), a combined analysis of these Rohrhofer 1993, Igersheim et al. 1994), and data, and also an analysis of our data in pollen investigations by e.g. Robbrecht and co- combination with the recently published rpsl6 workers (Robbrecht 1982, Jansen et al. 1996). data, rbcL/atpB-rbcL/rpsl6 (also referred to as The classification systems as well as the the 3-data matrix or analysis). As a result of taxonomic/morphological investigations have the phylogenetic analyses we present a new in their turn inspired phylogenetic investiga- classification of the subfamily Rubioideae. tions based on molecular data. In many of these, Rubioideae taxa have been included even if the focus has been on other groups or at Material and methods other taxonomic levels. Several studies have The sampling strategy in the phylogenetic analyses aimed at resolving the whole family phylogeny was to cover as many of the described tribes of the (Bremer and Jansen 1991, Bremer et al. 1995, subfamily Rubioideae as possible, and also to Bremer 1996a), others at specific tribes, e.g. include several taxa from large and diverse tribes. Rubieae (Manen etal. 1994, Manen and We did not manage to find material from two of the Natali 1995, Manen and Natali 1996, Natali twenty-eight tribes (Lathraeocarpeae and Pera- et al. 1996), and Psychotrieae (Nepokroeff meae) and for economical and technical reasons et al. 1999). These studies are based on mo- we did not sequence all taxa for both rbcL and the spacer. All 151 genera included in the resulting lecular markers designed to elucidate phylog- classification are listed in the appendices 1 and 2. enies at different taxonomic levels but the The outgroups were chosen to represent one results are congruent to a great extent. The member outside the Rubiaceae, results in some parts agree with classification Gelsemium (Gelsemiaceae; in the spacer and com- but in other parts they are totally different and bined analyses), and members of other subfamilies there are several important conclusions of the Rubiaceae (cf. Bremer 1996a), from concerning Rubioideae, e.g. that Hamelieae the subfamily , and and B. Bremer and J.-F. Manen: Rubioideae classification 45

Luculia (in the spacer analysis also were without indels and the alignments were simple and ) from the subfamily Cinchonoideae. and without any alternatives. In the analysis of our data in combination with the The parsimony analyses were done using rpsl6 data we used Gelsemium and Cinchona as PAUP 3.1 (Swofford 1993). Only phylogenetically outgroups. informative characters were included. The search Silica gel-dried or herbarium were used method for the combined matrices was heuristic in the DNA investigations. DNA was extracted, with 100 replications of RANDOM stepwise addi- amplified, and sequenced according to Bremer et al. tions of sequences, the TBR branch swapping, and (1995) and Backlund et al. (2000) for rbcL, and MULPARS options in effect. Support of the clades according to Manen et al. (1994) for the atpB-rbcL was calculated with bootstrap analyses with 1000 spacer. Table 1 provides a list of taxa included in replicates and TBR branch swapping and MUL- the phylogenetic analyses of rbcL and atpB-rbcL PARS off. data, with EMBL/GenBank accession number of the corresponding sequences, and with voucher information for new sequenced taxa. New rbcL Results sequences have been produced for 41 taxa and there The atpB-rbcL spacer matrix (63 taxa) com- are 52 new spacer sequences. All other atpB-rbcL prises 970 aligned positions, 453 of which are sequences used in these analyses have been pub- variable and 255 are potentially informative, 35 lished earlier by the first or second author alone or of which are indel characters. The matrix in collaboration with co-authors (Olmstead et al. rbcL 1993; Bremer et al. 1995; Bremer 1996a, b; Andrea- (106 taxa) comprises 1 402 sites, 560 of which sen and Bremer 1996; Manen et al. 1994; Manen are variable and 404 are potentially informa- and Natali 1995, 1996). For references to the rpsl6 tive. The combined rbcL/atpB-rbcL matrix (59 data see Andersson and Rova (1999). taxa) comprises 2 364 sites, 899 of which are The rbcL and atpB-rbcL spacer matrices com- variable and 533 are potentially informative. prise 106 and 63 taxa, respectively. The combined The combined rbcL/atpB-rbcL/rpsl 6 matrix (42 rbcL/atpB-rbcL matrix comprises 59 taxa. Most of taxa) comprises 3 254 aligned positions, 1 269 the taxa used in the combined matrix are of the of which are variable and 737 are potentially same species. However, , , informative, of which 57 are indel characters. , , , Praravinia, Ham- The atpB-rbeL spacer analysis (Fig. 1). The elia and Gardenia are represented by two distinct bootstrap analysis of the 63 spacer sequences species in the rbcL and in the atpB-rbcL matrix, respectively. In one analysis we included rpsl6 data (52 from Rubioideae and eleven outgroup from Anderson and Rova (1999) and performed a taxa) resulted in a tree illustrated in a simpli- combined analysis. In this 3-data matrix we fied form showing mainly the relationships of included 42 genera from the two studies. In several the tribes (Fig. 1). cases we had to compare different species from a In the atpB-rbcL spacer tree, based on 255 genus, but in genera suspected to be paraphyletic, phylogenetically informative characters, the e.g., and , we selected species mean bootstrap value for the nodes is 55%, that are supposed to be close and belong to the and 35% of the nodes have bootstrap values same monophyletic group. _>75%. The alignments of the atpB-rbcL spacer and the In agreement with earlier studies (Bremer matrices were first done by the Clustal rpsl6 et al. 1995, Natali et al. 1995, Andersson and program (Thompson et al. 1994) followed by Rova 1999) the tribes Hamelieae and Hillieae manual corrections. Some short regions could not be aligned and were excluded from the analyses. do not belong in the subfamily Rubioideae but Homologous indel events were coded as presence/ instead in the Cinchonoideae. (for- absence (1/0). When it was not possible to decide mer Virectarieae) is close to Gardenia of the for homology, the event was coded with a question- Ixoroideae (as in Bremer and Thulin 1998). mark. The aligned matrices are available on request The subfamily Rubioideae is well-supported or at http://www.unige.cjb.ch. The rbcL matrices with a bootstrap value of 100%. The base of the 46 B. Bremer and J.-F. Marten: Rubioideae classification

Table 1. List of taxa sequenced for rbcL and atpB-rbcL. Earlier unpublished sequences are indicated with a * and voucher information is given. All other taxa have been published earlier by the first or second author alone or in collaboration with co-authors (see Material and Methods). Names of herbaria are abbreviated according to Holmgren et al., 1990. All rps16 sequences analysed in this paper are from Andersson and Rova 1999 Accession EMBL/ Accession EMBL/ Source/ GenBank rbcL GenBank atpB-rbcL voucher information bojeri Z68787 (Hiern.) Klotzsch Blume sp. AJ002176 Amphiasma luzuloides AJ288594" , Herb. material, (K. Schum.) Bremek. Iversen et al. 87694 (UPS) Amphidasya ambigua Yl1844 (Standley) Standley Anthosperrnum X83623 AJ234028" Tanzania, Bremer 3093 (UPS) herbaceum L.f. Appunia guatemalensis AJ288593" AJ234009" North America, Herb. Donn. Smith material, Martinez 13581 (G) aristaum AJ288595" Ecuador, Bremer et al. Standley 3371 (UPS) hookeri King Z68788 AJ234032" , Wanntorp s.n. (S) laevigata L. X81092 Batopedina pulvinellata AJ288596" Zaire, Herb. material, E. Robbrecht Malaisse 7695 (UPS) glaberrima X83626 Engelm. Bouvardia glaberrima X76478 Engelm. Sond. sp. AJ288597" South , Bremer 3708 (UPS) glaucescens Z68789 (Hiern.) Verdc. Chasalia parviflora Benth. Z68790 Chazaliella abrupta Z68791 (Hiern) E. Petit & Verd. Cinchona pubescens Vahl X83630 Cinchona pubescens Vahl AJ233990" , McDowell. 4613 (DUKE) hirsutum X87145 (Bartling ex DC.) L,O. Williams Coccocypselum P. Br. sp. X811678 balansanum AJ288598" AJ234010" , Herb. Baill. material, Veillon 3765 (G) Cornmitheca liebrechtsiana AJ233999" , Herb. material, (De Wild & Th. Dur.) Vilks 478 (ME) Bremek, Conostomium quadrangulare Z68792 (Rendle) Cufod. B. Bremer and J.-F. Manen: Rubioideae classification 47

Table 1. (Continued) Accession EMBL/ Accession EMBL/ Source/ GenBank rbcL GenBank atpB-rbcL voucher information Coprosma ernodeoides AJ234029" , Kiehn 910114 (WU) A. Gray Hook. f. X87146 Coptosapelta diffusa AJ233987" , Herb. material, (Champ. ex Beneth.) Bartholomew et al. 1188 (ME) van Steenis Coussarea contracta AJ234007" Paraguay, Zardini and Beneth. & Hook. f. Velasquez 9774 (G) Coussarea macrophylla Y 11847 Muell. Arg. A J288629" AJ234011 * Zaire, Herb. material, brachynematum Hiern Lejoly 2707 (ME) glabra X81097 (L.) Ehrend. hymenodon AJ288599" , Herb. material, Hook. & Arn. Rodriguez 10 (K) Cruckshanksia hymenodon AJ234004" Chile, Herb. material, Billiet Hook. & Arn. and Jardin 5570 (ME) indicus Z68793 AJ234015" Cult., Missouri Bot. Gard. Gaertn. f. Bremer 3107 (UPS) xanthorrhoea Z68794 AJ234019" Tanzania, Bremer 3079 (UPS) (K. Schum.) Bremek. fruticosa Kuntze A J002177 alsinoides Z68795 (Chain. & Schlecht.) Standley Didymaea alsinoides AJ234036' Costa Rica, Kiehn sn (WU) (Cham. & Schlecht.) Standley sarmentosa SW. AJ288600" , Taylor 11749 (MO) litoraIis SW. AJ288601" AJ234025" , Kiehn sn (WU) Exostema caribaeum X83636 (Jacq.) Roem. & Schult. Exostema caribaeum A J233991' Cuba, Kiehn 910526 (WU) (Jacq.) Roem. & Schult. Faramea multiflora A. Rich. Z68796 Faramea porophylla AJ234008" Paraguay, Herb. material, Muell. Arg. Zardini 8630 (G) Retz. sp. AJ234012" , Herb. material, Malcomber 999 (BR, G, MO, TAN, WAG) Gaermera Retz. sp. Z68797 Gaillonia yemenensis AJ288630" Yemen,Thulin et al. 9365 (UPS) Thulin album Mill. X81090 circaeoides AJ288602" , Bremer Thunb. 3797 (UPS) 48 B. Bremer and J.-F. Manen: Rubioideae classification

Table 1. (Continued) Accession EMBL/ Accession EMBL/ Source/ GenBank rbcL GenBank atpB-rbcL voucher information Gardenia taitensis DC. AJ233988" Cult., Geneva Bot. Gard., Natali and Manen 007 (G) Gardenia thunbergia X83637 Linn. f. Gelsemium sempervirens Ait. L14397 AJ233985" repens Z68798 AJ234017" Cult., Uppsala Bot. Gard. (L.) I.M. Johnston Bremer 3130 (UPS) coriacea AJ288603" Cult., Bogor Bot. Gard., Blume Ridsdale XVII.C.103 (L) cuprea Griseb. X83641 Hamelia papillosa Urb. AJ233992" Jamaica, McDowell. 4600 (DUKE) Hedyotis caerulea AJ288604" Cult., Uppsala. Bot. Gard. Wight & Arn. Bremer s.n. (UPS) Hedyotis fruticosa Linn. Z68799 AJ234026" Hedyotis littoralis AJ288605" AJ234027" Cult., Vienna Bot. Gard., Kiehn (Hillebr.) Fosberg "" & Luegmayr 920823 (WU) Hedyotis nigricans AJ288606" USA, Miller et al., 8232 (MO) (Lain.) Fosberg Hemidiodia ocimifolia AJ288607" Ecuador, Bremer et al., 3340 Schum. (MO, QCA, QCNE, UPS) Hillia triflora X83642 AJ233993' Cult., Univ. of Colorodo, (Oerst.) C.M. Taylor Bremer 3101 (UPS) Hillia valeris Standley AJ288608" X81683 Costa Rica, Kiehn 880331 (WU) Hoffmannia refulgens X83644 Hemsl. x ghiesbreghtii Hemsl. formicarum X83645 Jack Hydnophytum formicarum X76480 Jack Hymenocoleus hirsutus AJ002178 (Benth.) Robbrecht platycarpa Arn. AJ288631" Sri Lanka, Herb. material, Lundqvist 11302 (UPS) caespitosa Z68800 Schnizl. pedunculatus Z68802 AJ234003" Tanzanaia, Andreasen 71 (UPS) E.A. Bruce Lelya prostrata (Good) AJ288609" , Herb. material, W.H. Lewis Thomson & Rawlins 5482 (K) Lerchea bracteata AJ288610" AJ233997" Sumatra, Herb. material, Valeton Axelius 343 (S) Luculia grandifolia X83648 AJ233986" Cult., Stockholm Univ., Ghose Bremer 2713 (S) Manettia bicolor Paxt. Z68803 B. Bremer and J.-F. Manen: Rubioideae classification 49

Table 1. (Continued) Accession EMBL/ Accession EMBL/ Source/ GenBank rbcL GenBank atpB-rbcL voucher information Manettia cordifolia AJ234022" , Herb. material, Mart. Novara 9814 (G) Mapouria cf. umbrosa Z68804 Muell. Arg. Maschalocorymbus AJ288611" Sabah, Ridsdale 2471 (L) corymbosus (Blume) Bremek. repens Linn. Z68805 Mitchella Linn. sp. AJ234016" Japan, Ehrendorfer 930905-1601 (WU) Mitracarpum villosum AJ288632" Sri Lanka, Herb. material, Cham. & Schlecht. Fagerlind 810 (S) citrifolia Linn. X83651 AJ234013" Cult., Uppsala Bot. Gard., Bremer 3106 (UPS) malayana Craib Z68806 AJ234033" Cult., Univ. of Aarhus, Larsen et al., 42486 (AAU, UPS) platyrea Becc. X87147 granadensis X83654 (L.F.) Druce zeylanicus Z68807 AJ233995" Sri Lanka, Bremer 937 Hook. (PDA, S, US) cf. X83655 corymbosa Linn. Oldenlandia goreensis Z68808 Sunamerhayes Oper cularia vaginata AJ234030" , Herb. material, Labill. Weber 9157 (G) vaginata Z68809 Labill. mungos Linn. X83656 Cult., Meisse Bot. Gard. Robbrecht s.n. (UPS) Ophiorrhiza Linn. sp. X81677 Sumatra, Frimmel s.n. (WU) Oreopolus glacialis AJ288612" Argentina, Swenson 328 (UPS) (Poepp. & Endl.) Ricardi Otiophora cupheoidea AJ288613" South Africa, Bremer N.E. Br. 3805 (UPS) oculata AJ288614" , Herb. material, S. Moore Puff 821222-2/1 (K) foetida Linn. AJ234006" Japan, Ehrendorfer 930830-0801 (WU) Aubl. sp. Z68810 Parapentas silvatica X83657 AJ234021" Tanzania, Bremer 3091 (UPS) (K. Schum.) Bremek. Paratriainia xerophila AJ288633" Madagascar, Herb. material, Bremek. Croat 30548 (MO) paucinervis Z68811 AJ233998" Tanzania, Bremer 3090 (UPS) (Hiern) Bremek. 50 B. Bremer and J.-F. Manen: Rubioideae classification

Table 1. (Continued) Accession EMBL/ Accession EMBL/ Source/ GenBank rbcL GenBank atpB-rbcL voucher information longituba Z68812 Oliver Pentanisia microphylla AJ234022" South Africa, Herb. Chior. material, Moll 610 (G) Pentanopsis fragrans Rendle Z68813 lanceolata X83659 Cult., Univ. of Connecticut, (Forsk.) Deft. Bremer 2702 (S) X76479 Geneva Bot. Gard., Natali (Forsk.) Deft. and Manen 004 (G) pentandrus X83660 AJ234024" Tanzania, Bremer 3082 (UPS) Vatke stylosa X81103 Beneth. & Hook. f. nobla Linn. Z68814 Linn. AJ234031" , Herb. material, Charpin and Rodriguez 18576 (G) Phylohydrax carnosa AJ288615" South Africa, Bremer (Hochst.) C. Puff 3783 (UPS) Placopoda virgata Z68815 Bolf. f. pendula Z68816 AJ234035" Canary Islands, Andreasen Ait. 1 (UPS) Praravinia densiflora Korth. AJ234000" Borneo, Clemens 33777 (G) Praravinia suberosa AJ288616" Sabah, no voucher, Ridsdale (Merill) Bremek. Pravinaria leucocarpa AJ288617" AJ234001" Borneo, Herb. material, Bremek. Collenette 21654 (G) labordei AJ234005" China, Herb. material, (Leveille) Merill apud Bartholomew et al. 2118 (ME) Rehder Prismatomeris beccarianum AJ288618" Sabah, Herb. material, (Baillon) J.T. Johansson Ridsdale 2461 (L) Psychotria kirlcii Hiern X83663 (bacteriophila) Yalet. Psychotria kirkii Hiern X76481 (bacteriophila) Yalet. Psychotria peteri Z68817 Verdcourt Z68818 AJ234018" Ecuador, Bremer et al. 3030 Muell. Arg. (NO, QCA, QCNE, UPS) Psychotria Linn. AJ002188 sp. " cephaelis" Psyllocarpus AJ288619" Brasil, Herb. laricoides Mart. material., Andersson et al. 355750 (UPS) B. Bremer and J.-F. Manen: Rubioideae classification 51

Table 1. (Continued) Accession EMBL/ Accession EMBL/ Source/ GenBank rbcL GenBank atpB-rbcL voucher information Putoria calabrica AJ288620" Herb. material, Jonsell (L.) DC. 4216 (UPS) Putoria calabrica X81672 Greece, Ehrendorfer (L.) DC. 930409-2301 (WU) Relbunium hypocarpium AJ288621" Cult., Meise Bot. Gard., Hemsl. Billiet 3788 (BR) pilosa Z68820 Ruiz. & Pav. tinctorum Linn. X83666 Linn. X76465 sessiliflora Standley AJ002186 Rutidea orientalis Z68862 D.M. Bridson Schenckia blumenaviensis AJ288622" Cult., Copenhagen Bot. K. Schum. Gard., Ryding 2359 (C) Schenckia blumenaviensis AJ233994" Brazil, Kiehn sn (WU) K. Schum. subandina Yl1859 AJ234014" Ecuador, Clark & Watt Krouse 783 (MO, QCNE, UPS) fetida Lam. Z68822 AJ234034" Cult., Univ. of Connecticut, Bremer 2717 (UPS) arvensis Linn. X81106 Spermacoce assurgens X81679 Ruiz &Pav. Spermacoce hispida Linn. AJ288623" Sri Lanka, Herb. material, Wanntorp et al. 2667 (S) Spermacoce laevis Roxb. Z68823 Spermadictyon suaveoIens Z68824 Roxb. Synaptantha tillaeacea AJ288624" Australia, Herb. material, (F. Muell.) J.D. Hook. Lazarides & Palmer 272 (K) cynocrambe X81680 Greece, Ehrendorfer Huth 930416-6201 (WU) TheIigonum cynocrambe X83668 Huth Triainolepis hildebrandtii AJ288625" AJ234020" Kenya, Herb. material, Vatke Bally 13258 (G) Trichostachys Hook f. in AJ288626" Cameroun, Sonk6 1725 (UPS) Beneth. & Hook. f. sp. ellipticum Thw. AJ288627" AJ234002" Herb. material, Lundqvist 11085 (UPS) muralis L. X81107 Virectaria major K. Schum. Yl1861 AJ233989" Herb. material, Reekmans 10916 (UPS) capitellatum AJ288628" AJ233996" Sabah, Ridsdale 2473 (L) Ridley 52 B. Bremer and J.-F. Manen: Rubioideae classification

GeIsemium-Gelsemiaceae 1 50 Coptosapelteae 2 93 Cinchonoideae incl. Hillieae and HameUeae 6 74 Ixoroideae incl. Virectaria 2 84 Ophiorrhizeae 4 100 UrophyUeae 5 Lt Lasiantheae 1 99 Coussareeae 4 Craterispermeae 1 Schradereae 1 Morinda 1 Appunia 1 99 99 Coelospermum 1 Gaermereae 1 --1065r---~0 group 2 J Morindeae-Mitchella Prismatomeris 1 =~ 59 Psychotrieae 4 5__~6 Psychotria alliance Danaideae 1 98 Spermacoceae-Pentas group 4 ~. 96 Spermacoceae-Hedyotis group 7 1 97 -- Coprosma 1 Opercularia 1 Phyllis 1 98 Argostemmateae 2 Spermacoceae alliance 95

83 8~100 PlocamaPaederieaep.p. 1 2 89 Putoria 1 Theligoneae 1 Rubieae 2 Fig. 1. Simplified illustrating the relationships of Rubioideae based on atpB-rbcL sequences (analysis includes 52 ingroup taxa). Numbers above nodes indicate bootstrap values (___50%) from 1000 replicates with TBR branch swapping, all bootstrap values _>50% are indicated. Numbers behind names represent number of investigated taxa on this branch

Rubioideae forms a trichotomy. One branch is supported as monophyletic). The other branch the Ophiorrhizeae (bootstrap 84%), another is (bootstrap 97%) is here called the Spermaco- the Urophylleae (bootstrap 100%), and the ceae alliance and contains Spermacoceae (not third branch includes the remaining part of the supported as monophyletic), Anthospermeae subfamily (bootstrap 98%). In this large clade, (not supported as monophyletic), Argostemma- Lasianthus (Lasiantheae) is the first taxon to teae, Danaideae, Paederieae (in this analysis branch off followed by the clade Coussareeae paraphyletic), Rubieae, and Theligoneae. (bootstrap 99 %, including Coussarea, Fararnea, The rbcL analysis (Fig. 2). The bootstrap Cruckshanksia, former Hedyotideae/Crucks- analysis of the 106 rbcL sequences resulted hanksieae and Coccocypselum). The sister in a tree illustrated in a simplified form group to Coussareeae is not well-supported showing mainly the relationships of the tribes (56%). However, within this clade there are two (Fig. 2). well-supported subclades. One of them here The topology of this rbcL tree (of 102 called the Psychotrieae alliance (bootstrap Rubioideae taxa and four outgroup taxa) is 99%) contains Pychotrieae, Craterispermeae, similar to the spacer tree (Fig. 1) but the support Gaertnereae, Schradereae, and Morindeae (not values are slightly higher. In the rbcL tree, based B. Bremer and J.-F. Manen: Rubioideae classification 53

Gelsemium-Gelsemiaceae 1 spacer and rbcL trees. The main differences are Luculia 1 Cinchona 1 the better resolution within the trees and much Gardenia 1 higher support values for many branches. In i 60 Ophiorrhizeae 4 this combined tree, based on 533 phylogenet- 100 Urophylleae 5 ically informative characters, the mean I I 1oo Lasiantheae 2 Coussareeae 6 bootstrap value for the nodes is 81%, and Craterispenneae 1 68% of the nodes have bootstrap values ___75%. Gaertnereae i The Rubioideae clade is supported by Schradereae 1 100%. The most basal clade to branch off Morindeae-Morinda group 3 within the Rubioideae is the Ophiorrhizeae Morindeae-Mitchella group 2 Prismatomeris 1 (bootstrap 92%; including Ophiorrhiza, Psyehotrieae 14 Lerchea, Neurocalyx, and Xantophyturn), sepa- Danaideae 1 Psychotria alliance [ rated (87%) from the following Urophylleae Spermacoceae-Pentas group 11 (bootstrap 100%; including Urophyllum, Spermacoceae-Hedyotis group 26 Anthospermeae 7 Pravinaria, Praravinia, and Pauridiantha). Argostemmateae 2 High bootstrap values (97%) support the Paederieae p.p. 2 isolation of Lasianthus from the basal clades Paederieae p.p. 3 Spermacoceae alliance Theligoneae 1 and the remaining taxa (100%). The sister Rubieae 9 group of Lasianthus comprises three main branches. The relationships between these are Fig. 2. Simplified phylogenetic tree illustrating the not supported (<50%). The three branches are relationships of Rubioideae based on rbcL sequences the Coussareeae (bootstrap 100%, including (analysis includes 102 ingroup taxa). Numbers above nodes indicate bootstrap values (___50%) from 1000 here also Coccocypselum), the Psychotrieae replicates with TBR branch swapping, all bootstrap alliance (bootstrap 99%), and the Sperma- values >_50% are indicated. Numbers behind names coceae alliance (bootstrap 100%). Inside the represent number of investigated taxa on this branch Psychotrieae alliance clade, the tribe Crateri- spermeae is sister to the remaining part (boot- on 404 phylogenetically informative characters, strap 54%), which comprises Gaertnereae, the mean bootstrap value for the nodes is 60%, Morindeae, Schradereae, and Psychotrieae. and 38% of the nodes have bootstrap values The relationships between these ,tribes are not _>75%. All tribes that are supported in the spacer supported (<50), and of these tribes only two tree are also supported in the rbcL tree. Further, are represented by several taxa of which only in the rbcL tree there is also support (74%) for a tribe Psychotrieae has high support (83%). monophyletic Anthospermeae and a monophy- Inside the Spermacoceae alliance clade, the letic Spermacoceae (99%), which are not sup- position of Danaideae has low support (boot- ported in the spacer tree. strap 51%). The remaining taxa are included The combined rbcL[atpB-rbcL analysis in two clades, one is the Spermacoceae (boot- (Fig. 3). The analysis of 59 taxa (49 Rubioi- strap 99%) which are clearly divided in two: deae taxa and ten outgroup taxa) from a one subclade (bootstrap 100%) containing the combined matrix of rbcL and the spacer Pentas group and former members of Knox- sequences resulted in two trees 1 787 steps ieae and Triainolepideae, and another sub- long with a consistency index of 0.427 (exclud- clade (bootstrap 100%) containing e.g. the ing uninformative characters) and a retention Spermacoce and the Oldenlandia groups. The index of 0.713. The strict consensus tree with other main branch of the Spermacoceae alli- bootstrap values indicated is presented in ance is not well-supported (54%) but includes Fig. 3. the relatively well-supported clades of Antho- The analysis of the combined data pro- spermeae (bootstrap 100%), Argostemmateae duced trees very similar to the topology of the (bootstrap 100%), and Paederieae, Theligo- 54 B. Bremer and J.-F. Manen: Rubioideae classification

Gelsemium Luculia Gardenia Virectaria Cinchona Exostema HilIia val. Hillia tri. Hamelia Schenckia Neurocalyx Xanthophytum OPH Lerchea Ophiorrhiza Pravinaria Praravinia URO Pauridiamha Urophyllum Lasianthus LAS Coccocypselum ! Coussarea COU Faramea Craterispermum CRA Appunia Coelospermum Morinda MOR Prismatomeris Damnacanthus Mitchella Gaertnera GAE Schradera SCH Geophila Psychotria poe, l-lydnophytum PSY Psychotria kit. Danais DAN Parapentas Pentas

P entanisia t,,~. Triainolepis Hedyotis fru. Pentodon Ernodea SPE Hedyotis lit. Manettia Bouvardia Spermacoce Anthospermum Phyllis ANT Coprosma Opercularia Argostemma ARG Mycetia Serissa Plocama PAE Putoria Theligonum THE Didy. maea Rubia RUB B. Bremer and J.-F. Manen: Rubioideae classification 55 neae, and Rubieae (bootstrap 78%). those genera that we have sequenced and genera Paederieae are paraphyletic. with a close relationship to sequenced genera as The combination of our rbcL]atpB-rbcL judged from morphology. data with rpsl6 data (Fig. 4). The analysis of 42 taxa (38 Rubioideae taxa and four outgroup Subfamily Rubioideae Verdcourt, Bull. Jard. taxa) from a combined matrix of data from the Bot. lEtat Brux. 28:280 (1958) rbcL, the atpB-rbcL spacer and the rps16 - Cephaelidoideae Rafinesque, Ann. G6n. intron (Anderson and Rova 1999) resulted in Sci. Phys. Brux. 6:86 (1820) 24 trees 2 096 steps long with a consistency - Richardioideae Rafinesque, Ann. G6n. index of 0.503 (excluding uninformative char- Sci. Phys. Brux. 6:84 (1820) acters) and a retention index of 0.682. The - Spermacocoideae Chevall., F1. G6n. Env. strict consensus tree with bootstrap values Paris 2:605 (1827) as "Spermacocceae" indicated is presented in Fig. 4. - Anthospermoideae Kostel., Allg. Med.- The topology of the consensus tree is Pharm. F1. 2:537 (1833) as "Anthospermeae" almost identical or congruent with the rbcL/ - Hedyotidoideae Kostel., Allg. Med.- atpB-rbcL tree but the support value for each Pharm. F1. 2: 538, 569 (1833) as "Hedyoti- branch is higher, as could be expected since deae" more characters are included. For example, the - Opercularioideae Kostel., Allg. Med.- separation between the Coussareeae and Pharm. F1. 2:537 (1833) as "Opercularieae" Psychotrieae and Spermacoceae alliances are - Paederioideae Kostel., Allg. Med.- better supported (70% compared to <50%), Pharm. F1. 2: 537, 564 (1833) as "Paederieae" and the Anthospermeae, Argostemmateae, - Gaertneroideae Arn., Pug. P1. Ind. Or.: Paederieae, Theligoneae, and Rubieae branch 351,352 (1836) as "Gaertnerieae" has higher support (91% compared to 54%). - Coccocypseloideae Burmeist., Handb. This 3-data tree, based on 737 phylogenetically Naturgesch., 286 (1837) as "Coccocypseleae" informative characters, has a mean bootstrap - Manettioideae Burmeist., Handb. Na- value for the nodes of 81%, and 72% of the turyesch., 286 (1837) as "Manettieae" nodes have bootstrap values >_75%. - Aparinoideae Pfeiff., Nomencl. Bot. 1(1): Two minor differences between this tree 320 (1872) and the combined rbcL/atpB-rbcL tree are that the trichotomy of Ophiorrhizeae, Urophylleae - Pomazotoideae Bremekamp ex S. Dar- and the rest of the subfamily is unresolved, and win, Taxon 25:605 (1976) that the relationships within the Psychotrieae , herbs, or less common trees. Rap- are less resolved, but these relationship are not hides present. Heterostylous common. supported in the rbcL/atpB-rbcL (<50%) tree. Stipules entire, bifid or often fimbriate. Corolla Classification. As a result of the phyloge- aestivation always valvate. Ovary 1-12-1ocular, netic analyses of molecular data in combination most often 2-1ocular either with many or with with morphological characters and comprehen- single ovules in each locule, more rarely two or sive information from literature we here present few in each locule. Fruits dry or fleshy, a revised classification of the subfamily Rubioi- dehiscent or indehiscent. deae (below and Table 2). We have only listed

Fig. 3. Strict consensus tree of two equally parsimonious trees of Rubioideae based on rbcL and atpB-rbcL sequences, after a heuristic search with 100 replicates and TBR branch swapping. Numbers above nodes indicate bootstrap values (>50%) of 1000 replicates with TBR branch swapping, all bootstrap values >50% are indicated, Vertical bars and corresponding letters (first three letters of the tribes cf. Table 2) represent the tribal classification according to Brerner and Manen 56 B. Bremer and J.-F. Marten: Rubioideae classification

Gelsemium Cinchona Hamelia Hillia vaL Ophiorrhiza OPH Pauridiantha URO Urophyllum

Lasianthus LAS Oreopolus Coccocypselum

Declieuxia COU Coussarea Faramea

aaertnera GAE

Schradera SCH Hydnophytum

Geophila PSY Psychotria poe. Coelospe rmurn Morinda MOR C~ Damnacanthus Mitchella Pentanisia Psychotria alliance Pentas r,,d, Agathisanthemum Pentodon Hedyotis fru, SPE Ernodea Bouvardia Manettia Sperrnacoce Phyllis Spermacoceae alliance Coprosma ANT Opercularia Argostemma ARG Mycetia Serissa I I Plocama I PAE f Putoria I Theligonum I THE Didymaea I RUB Rubia B. Bremer and J.-F. Manen: Rubioideae classification 57

Tribes included (here investigated): Antho- Genera included (here investigated): spermeae, Argostemmateae, Coussareeae, Urophyllum, Amphidasya, Comrnitheca, Craterispermeae, Danaideae, Gaertnereae, Maschalocorymus, Praravinia, Pravinaria, Pau- Lasiantheae, Morindeae, Ophiorrhizeae, Pae- ridiantha. derieae, Psychotrieae, Rubieae, Schradereae, Urophyllum and Pauridiantha both have Spermacoceae, Theligoneae, Urophylleae. the rare basic number x = 9 and they also have Uncertain status (not investigated): Lath- bimodal karyotypes (Kiehn: pers. comm.). In raeocarpeae, Perameae. Andersson and Rova (1999) Raritebe belongs to the Urophylleae clade. Raritebe was earlier Tribe Ophiorrhizeae Bremekamp ex Verdcourt, (Robbrecht 1993) placed in the Isertieae Bull. Jard. Bot. l~tat Brux. 28:281 (1958) (subfamily Cinchonoideae) and reported not - Pomazoteae Bremekamp ex S. Darwin, to contain raphides. We have not seen any Taxon 25:605 (1976) material of the genus but if the position is Useful recent studies: Darwin (1976), correct it is the first report of a genus without Bremer (1979), Axelius (1987, 1990) raphides in Rubioideae. Herbs or subshrubs to small trees. Stipules entire, bifid to fimbriate. Flowers homo- or Tribe Lasiantheae B. Bremer and Manen, trib. heterostylous, Neurocalyx with adnate anthers. nov. Ovary 2-1ocular, with many ovules. Fruits dry, - Dressleriopsideae Dwyer, Ann. Missouri. dehiscent (flattened capsules in Ophiorrhiza) or Bot. Gard. 67:11 (1980)- not validly published indehiscent with thin to thick pericarps, with Type genus: Lasianthus many small (dust) seeds. Pollen 3-colporate. Frutices, arbores parvae vel suffrutices. Chromosome basic number x = 11 (12) with 2- Stipulae integrae saepe triangulares. Flores or 4-ploidy level (Kiehn 1995). interdum heterostyli. Ovaria 2- vel 4-12-1ocu- Genera included (here investigated): Oph- laria, ovulo in quoque loculo singulari. iorrhiza, Neurocalyx, Lerchea, Xanthophytum. Fructus drupacei, nec compositi nec aggregati, Inclusion based on morphology: Copto- saepe cyanei ad ateri pyrenis 2 vel 4-12. - phyllum (incl. Pomazota), Spiradiclis, (and Differt a tribus affinibus fructibus cyaneis vel included in Xanthophytum: Paedicalyx and atris, pyrenis 4-12 (Trichostachys 2) et Xanthophytopsis). inflorescentiis saepe axillaribus sessilibus. Shrubs, small trees, or subshrubs. Stipules entire, often triangular. Flowers sometimes Tribe Uropbylleae Bremekamp ex Verdcourt, Bull. Jard. Bot. l~tat Brux. 28:281 (1958) heteostylous. Ovary 2- (Trichostachys) or 4-12- 1ocular, with a single ovule in each locule, erect - Pauridiantheae Bremekamp ex S. Dar- win, Taxon 25:605 (1976) from the base. Fruits drupaceous, not com- Shrubs, subshrubs, or small trees. Stipules pound or aggregated, often blue to black, with entire or fringed. Ovary 2-pluri-locular. Fruits 2 or 4-12 pyrenes. Chromosome basic number fleshy with many seeds. Flowers unisexual or x= 11 with 4- (Bir et al. 1984) or 22-ploidy hermaphroditic, homo- or heterostylous. Pol- level (Kiehn pers. comm.). len 3-colporate. Chromosome basic number Genera included (here investigated): x = 9 with 2- or 6-ploidy level (Kiehn 1995). Lasianthus, Trichostachys.

Fig. 4. Strict consensus tree of 24 equally parsimonious trees of Rubioideae based on rbcL, atpB-rbcL, and rps16 sequences, after a heuristic search with 100 replicates and TBR branch swapping. Numbers above nodes indicate bootstrap values (_>50%) of 1 000 replicates with TBR branch swapping, all bootstrap values _>50% are indicated. Vertical bars and corresponding letters (first three letters of the tribes cf. Table 2) represent the tribal classification according to Bremer and Manen 58 B. Bremer and J.-F. Manen: Rubioideae classification

Table 2. Tribes included in or associated with the subfamily Rubioideae (Rubiaceae) Tribes included in or associated with the Rubioideae by Bremekamp (1952, 1954, 1966), Verdcourt 1958, 1975), or Robbrecht (1988, 1993) and compared to the classification of Bremer and Manen. All investigated and accepted Rubioideae tribes are in boldface. Under the authors, tribal names are indicated with the first three letters of the tribal names; subfamilies are indicated with the first four letters of subfamilial names ANTIrheoideae, CINChonoideae, HILLioideae, POMAzotoideae, UROPhylloideae. "x": the tribe is ac- cepted in the subfamily Rubioideae; "?": uncertain position according to the author; "in": the tribe is included in another tribe

Bremekamp Verdcourt Robbrecht Bremer and Manen

Tribes of Rubioideae Anthospermeae x x X X Argostemmateae x x X X Coussareeae x x x new cirumscription Craterispermeae x x ANTI x Danaideae new tribe Gaertnereae x in PSY? in PSY x Lasiantheae new tribe Morindeae x x x x Ophiorrhizeae UROP x x new circumscription Paederieae x x x paraphyletic? Psycbotrieae x x x x Rubieae x x x x Schradereae x x x x Spermaeoeeae x x x x Theligoneae x x Urophylleae UROP x CINC x Included in other tribes of Rubioideae Coccocypseleae x x x in COU Cruckshanksieae x x in HED in COU Hedyotideae x x x in SPE Knoxieae x x ANTI in SPE Manettieae in HED in HED ? in HED/CIN in SPE Opercularieae ? in ANT in ANT Pauridiantheae UROP in URO CINC in URO Pomazoteae POMA ? in HED in OPH Triainolepideae x in PSY x in SPE Included in other Subfamilies Hamelieae x x x CINC Hillieae HILL ?x CINC CINC Virectarieae in OPH CINC in HED IXOR Incertae sedis Lathraeocarpeae x x ? no material Perameae x x ? no material

Inclusion based on morphology (cf. Puff Tribe Coussareeae J. D. Hooker, in G. Ben- and Igersheim 1994): Metabolus (incl. Alla- tham and J. D. Hooker, Genera Plantarum 2: eophania). 9, 24 (1873) B. Bremer and J.-F. Manen: Rubioideae classification 59

- Coccocypseleae Bremekamp, Rec. Tray. Genus included (here investigated): Crat- Bot. Need. 3l: 253 (1934) erispermum. - Cruckshanksieae J. D. Hooker Genera Plantarum 2: 9, 20 (1873) Tribe Gaertnereae Bremekamp ex S. Darwin, Useful recent studies: Dwyer (1966), Kirk- Taxon 25:601 (1976) bride (1976), Taylor (1996a) Useful recent studies: Igersheim et al. Herbs (creeping in Coccocypselum), sub- (1994), Jansen et al. (1996) shrubs, shrubs, or small trees. Stipules entire, Shrubs or trees. Stipules entire and united cleft, as a ridge with appendages, or deeply into a long cylindrical sheath, often with setae. bilobed. Flowers homo- or heterostylous, Flowers often small and white. Ovary superior, often 4-merous, white, blue, or bright yellow 2-1ocular, with single erect ovule in each locule. (Cruckshanksia, Oreopolus). Ovary 1-2-1ocu- Fruits fleshy, with one-seeded pyrenes. Pollen lar, with 1-2 or many (CoccocypseIum) ovules 2-3-colporate. per locule. Fruits often flattened (not Coc- Genus included (here investigated): Gaert- cocypselum), fleshy, white (Coussarea) or nera. often blue (Faramea, Coccocypselum) berries, Inclusion based on morphology (cf. Igers- schizocarps (Declieuxia), or thin-walled heim et al. 1994, and Jansen et al. 1996): capsules ( Cruckshanksia, Oreopolus), with . 1-2 or many seeds, often flattened. Pollen 3-colporate or 2-4-porate. Chromosome basic Tribe Morindeae Miquel, Flora van Neder- number x = 10, 11? with 2- or 4-ploidy level landsch Indie 2: 239, 241 (1857) (Kiehn 1995). Useful recent study: Igersheim and Robbr- Genera included (here investigated): Couss- echt (1993) area, Coccocypselum, Declieuxia, Cruckshank- sia, Faramea, Oreopolus. - Subtribe Morindinae de Candolle, Prodro- In Andersson and Rova (1999) and mus Systematis Naturalis 4: 342, 446 (1830) Heterophyllaea are nested within our Cous- Useful recent studies: Johansson (1988, sareeae. We have not had the possibility to 1994) sequence these genera, but Hindsia shows at Shrubs, small trees, or lianas. Stipules least macromorphological similarity to Decli- entire or dentate, usually connate to sheath- euxia. If this position is correct the fruit ing. Flowers usually not heterostylous. Ovary morphology of the tribe is even more variable 2-1ocular, with 2 erect ovules in each locule, as the fruits of Hindsia are capsular with hard later subdivided by secondary septum. Fruits walls and winged seeds. drupaceous, often aggregated. Pollen 3- 4-colporate. Chromosome basic number Tribe Craterisperlneae Verdcourt, Bull. Jard. x=ll with 2-, 4-, 8-, or 20-ploidy level Bot. l~tat Brux. 28:281 (1958) (Kiehn 1995). Useful recent study: Igersheim (1992) Genera included (here investigated): Mo- Shrubs or trees. Stipules entire. Inflores- rinda, Appunia, Coelospermum, Gynochthodes. cences axillary and condensed and borne on a Inclusion based on morphology: Pogono- stout short peduncle. Flowers heterostylous. lobus. Ovary 2-1ocular, with a single pendulous ovule in each cell (one aborted?). Fruits fleshy - Subtribe Prismatomerinae Ruang, Acta (thickness of endocarp varies) with one seed. Phytotaxon. Sin. 26:446 (1988) Pollen 3-colporate. Chromosome basic num- Useful recent studies: Johansson (1987A, ber x = l 1 with 2-ploidy level (Kiehn 1995). 1987B, 1989), Robbrecht et al. (1991) 60 B. Bremer and J.-F. Manen: Rubioideae classification

Shrubs or trees. Stipules entire or often This tribe usually includes many genera bilobed. often heterostylous. Ovary 2- (e.g. Robbrecht 1988) and Psychotria has been locular, with a single ovule in each locule. shown to be highly paraphyletic and many of Fruits fleshy berries, often dark purple or blue, the described genera are nested within it. free or fused. Pollen 3-5-colporate. Major revision of the whole tribe with new Genus included (here investigated): Pris- circumscriptions of genera will probably matomeris. follow from initiated phylogenetic analysis Inclusion based on morphology: Renellia (e.g. Nepokroeff pers. comm). Andersson and (incl. Didymoecium), Motleyia. Rova (1999) have shown that Readea, Strebl- osa and Margaretopsis belong to this group of - The Mitchella group taxa but we have not sequenced these genera Useful recent study: Robbrecht etal. and it is not obvious from the morphology that (1991) they belong here. Thorny shrubs or creeping herbs. Stipules entire or rarely bilobed. Flowers homo- Tribe Sehradereae Bremekamp, Rec. Trav. (Damnacanthus) or hetero-stylous (Mitchella). Bot. Neerl. 31:253 (1934) Ovary 2-1ocular, with one pendulous ovule in Useful recent studies: Puff et al. (1993), each locule. Fruits fleshy, free or fused in pairs. Puff and Buchner (1998), Puff et al. (1998a), Pollen 3-6-colporate. Chromosome basic num- Puff et al. (1998b) ber x---11 with 2-ploidy level (Robbrecht, or often epiphytic climbers with et al. 1991). : adhesive roots. Stipules entire. Ovary 2-(3-4)- Genera included (here investigated): Mitch- locular. Flowers in congested spherical inflo- ella, Damnacanthus. rescences, heterostyly has been reported. Fruits fleshy, baccate, with many seeds. Chromosome Tribe Psyehotrieae Cham. and Schltdl., Lin- basic number x = 11 with 2-ploidy level (Kiehn naea 4: 4. 1829 1995). Pollen 2-3-(4-)-porate. - Psathureae A. Rich. ex Dumort., Anal. Genera included (here investigated): Schra- Fam. PI.: 32. 1829 dera. Useful recent treatment: Taylor (1996b), Inclusion based on morphology (cf. Puff Nepokroeff et al. (1999) et al. 1993): Lecananthus, Leucocodon. Shrubs, trees or herbs, or epiphytes. Stip- ules divided or rarely entire. Flowers often Tribe Danaideae B. Bremer and Manen, trib. small, white and heterostylous. Ovary often 2- nov. locular, with single erect ovule in each locule. Type genus: Dana& Fruits fleshy, with one-seeded pyrenes. Seeds Useful recent studies: Buchner and Puff often with horny endosperm. Pollen very (1993), Puff and Buchner (1994) variable, 0-5-aperturate. Chromosome basic Lianae lignosae, frutices interdum scan- number x=(10), 11 with 2-12-ploidy level dentes vel arbores parvae. Stipulae integrae, (Kiehn 1995). bifidae vel fimbriatae. Flores heterostyli. Co- Genera included (here investigated, an * rollae valvatae vel reduplicatae-valvatae. indicates a paraphyletic taxon): Psycho- Ovaria 2-1ocularia in quoque loculo ovulis tria*, Amaracarpus, Cephaelis, Chasallia, numerosis. Fructus capsulares loculicidi Chazaliella, Geophila, Hydnophytum, Hymeno- vel septicidales seminibus alatis. Pollen 3-4 coleus, Myrmecodia, Palicourea*, Rudgea, (-5)-colporatum. - Differt a tribuluis ceteris Uragoga. habitibus plerumque scandentibus lignosis et Inclusion based on morphology: seminibus alatis (itidem in Bouvadria et Manet- Anthorrhiza, Myrmephytum, Squamellaria. tia; Spermacoceae). B. Bremer and J.-F. Manen: Rubioideae classification 61

Woody lianas, climbing shrubs, shrubs, or Kadua), Hemidiodia, Knoxia, Kohautia, Lelya, small trees. Flowers heterostylous. Stipules en- Manettia, , Oldenlandia*, Otiopho- tire, bifid or fimbriate. Corolla valvate or val- ra, Otomeria, Parapentas, Paratriaina, Penta- vate-reduplicate (Danais and Schismatoclada). nisia, Pentanopsis, Pentas, Pentodon, Ovary 2-1ocular with numerous ovules Phylohydrax, Placopoda, Psyllocarpus, Richar- in each locule. Fruits capsular loculicidal dia, Triainolepis. (Danais and ) or septicidal (Schismato- A majority of the former Hedyotideae, clada), with winged seeds. Pollen 3-4(-5)-colpo- Knoxieae, and Spermacoceae s.s. genera rate. should be included in this tribe. Inclusion Genus included (here investigated): Danais. based on morphology is supported for at least Inclusion based on morphology: (cf. Buch- the following genera: Chaemepentas, , ner and Puff 1993) SchismatocIada (inclusion , Dibrachionostylus, Dolichometra, He- supported by unpublished rbcL data: Bremer), dythyrsus, , Lucya, Manostachya, Payera. Mitrasacmopsis, Neohymenopogon, Nodoca- paea, Pseudohedyotis, Schwendera, Synaptan- Tribe Spermaeoeeae Bercht. and J. Presl, Prir. tha, Staelia, Thecorchus, Thyridocalyx. Rostlin: 256 (1820) - the change of the authors from earlier A. Rich. ex Dumort., Analyse des Tribe Anthospermeae Chamisso and Schlech- familles des plantes: 33 (1829) was first noted tendal ex de Candolle, Prodromus Systematis by J. Reveal (pers. comm.) Naturalis 4: 343, 578 (1830)

- Hedyotideae Chamisso and Schlechten- - Opercularieae A. Rich. ex de Candolle, dal ex de Candolle, Prodromus Systematis Prodromus Systematis Naturalis 4: 343, 614 Naturalis 4: 342, 401(1830) (1830) - Knoxieae Hooker f. in Bentham and - Durringtonieae Henderson and Guymer, Hooker, Genera plantarum 2: 9, 21 (1873) Kew Bull. 40:97-107 (1985) - Manettieae Bremekamp, Rec. Tray. Bot. Useful recent studies: Puff (1982, 1986), Neerl. 31:253 (1934) Robbrecht (1982) - Triainolepideae Bremekamp, Proc. Kon. Herbs, shrubs, dwarf shrubs, or small Akad. Wetensch. 59:3 (1956) trees. Stipules entire or divided. Flowers Useful recent studies: Puff (1988), Puff and usually unisexual, anemophilous, not hetero- Robbrecht (1989), Mena (1990) stylous. Stamens usually inserted low in the Herbs or subshrubs (a few species are corolla. Stigma long and filiform. Ovary 1-2- small trees). Stipules fimbriate. Flowers quite (5-)locular, with a single erect ovule in each often heterostylous. Ovary 1-5- or often 2- locule. Fruits dry and splitting into cocci or locular, with many to single ovules in each capsular, or fleshy. Chromosome basic locule. Fruits dry, dehiscent or indehishent, or number x= ll (one exception of 10) with rarely fleshy (e.g. Triainolepis) with many to 2-20-ploidy levels (Kiehn 1995). Pollen 3- single seeds. Pollen often 3-colporate-pluricol- colporate. pate. Chromosome basic number very vari- Genera included (here investigated): An- able x=6-17 with 2-20-ploidy levels (Kiehn thospermum, Carpacoce, Coprosma, Galopina, 1995). , Nertera, Opercularia, Phyllis. Genera included (here investigated, an * Inclusion based on morphology: Durring- indicates a paraphyletic taxon): Spermac- tonia, Eleutheranthus, (incl. Co- oce* (incl. Borreria), Agathisanthemum, rynula), , Peratanthe, Amphiasma, Arcytophyllum, Batopedina Bou- (inclusion of Pomax supported by unpublished vardia, Carphalea (incl. Dirichletia), Conosto- rbcL data Bremer). mium, Diodia, Ernodea, Hedyotis* (incl. 62 B. Bremer and J.-F. Manen: Rubioideae classification

Tribe Argostemmateae Bremekamp ex Verd- one ovule. Fruits fleshy, with elaiosome and a court, Bull. Jard. Bot. l~tat Brux. 28:281 single seed. Pollen (3-)4-8-porate. Chromo- (1958) some basic number x= 10, 11 with 2-ploidy Useful recent study: Bremer (1989) level (Kiehn 1995). Herbs with iso- or anisophyllous leaves. Genus included (here investigated): Theli- Stipules entire or slightly cleft. Flowers her- gonum. maphroditic. Stamens usually inserted at base of the corolla, adnate into an anther cone or Tribe Rubieae Baill., Hist. P1. 7: 365, 390 free. Anthers open with vertical slits or rarely (1880) with pores. Ovary 2-6-1ocular. Fruit a succulent - Galieae A. Rich. ex Dumort., Anal. Faro. opening by an apical operculum PI.: 33. (1829) (Argostemma) or a berry (Mycetia), with many - Asperuleae A. Rich., M6m. Soc. Hist. small seeds. Chromosome basic number x = 11, Nat. Paris 5:126 (1830) 14 with 2- or 4-ploidy levels (Kiehn 1995). Useful recent studies: Manen et al. (1994), Genera included (here investigated): Argo- Natali et al. (1996) stemma, Mycetia. Herbs or rarely subshrubs, with polygonal stems. Leaves (and or -like stipules) verticil- late. Flowers generally perfect and heterosty- Tribe Paederieae de Candolle, Prodromus lous, Phuopsis with secondary pollen Systematis Naturalis 4: 342, 470 (1830) presentation. Calyx rudimentary. Ovary 2-1oc- - Putorieae de Candolle ex Sweet, Sweet's ular, with single ovule in each locule. Fruits dry Hortus Britannicus, ed 3, 325 (1839) or fleshy, didymous, seeds usually adhering to Useful recent studies: Puff (1982), Thulin the pericarp. Pollen pluricolpate. Chromosome (1997) basic number x = 9-12 with 2-12-ploidy levels. Shrubs, climbers, dwarf shrubs and herbs, Genera included (here investigated, an many with a foetid smell. Stipules divided or * indicates a paraphyletic taxon): Rubia, entire. Flowers often heterostylous, Stamens Asperula*, Galium*, Relbunium, Didymaea, inserted in upper part of corolla, Ovary 2-5- Cruciata, Phuopsis, Sherardia, Valantia. locular, with a single erect ovule in each locule. Inclusion based on morphology: , Fruits dry, dehishent into mericarps or open- , Mericarpaea (inclusion supported ing with operculum, or fleshy, indehiscent. by unpublished atpB-rbcL data: Manen), Mi- Chromosome basic number x = 11 (rarely 10, crophysa, Warburgia. 12, 13) with 2-, 4-, 6- or 8-ploidy levels (Kiehn 1995). Pollen 3-colpate. Genera included (here investigated): Pae- Discussion deria, Gaillonia, Plocama, Putoria, Serissa, The power of the present study is that the Spermadictyon. phylogeny and proposed classification is based Inclusion based on morphology: Choulet- on many characters from different molecular tia, Jaubertia, , , Pseu- markers. One is protein-coding (rbcL), one is a dogaillonia, Pseudopyxis, Pterogaillonia. non-coding spacer (the atpB-rbcL spacer), and the third is a non-coding intron marker (rpsl6 Tribe Theligoneae Wunderlich ex S. Darwin, from Andersson and Rova 1999). Taxon 25:607 (1976) Another strength of our study is the Useful recent study: Wunderlich (1971) comprehensive tribal sampling, e.g., we in- Herbs with upper leaves alternate, aniso- clude, for the first time, representatives of the phyllous. Stipules sheathing. Flowers unisexu- tribe Craterispermeae and the former tribes al, anemophilous. Male flowers usually with Pomazoteae and Triainolepideae. Our an- many, up to 30 stamens. Ovary 1-1ocular, with alysed taxa represent all except two of the 28 B. Bremer and J.-F. Manen: Rubioideae classification 63

Rubiaceae tribes that have been associated from our combined analysis including their with the subfamily Rubioideae. We accept 16 rpsl6 data are different from their results. in Rubioideae (Table 2), nine are included as However, in our analysis the rpsl6 data are a synonyms, three are included in other subfam- minor part of the matrix. Other explanations ilies and two have been left "incertae sedis" as might be that we use different alignments, or we did not manage to get any material. If different ways of analysis. They never com- compared to earlier classifications of the sub- pleted their analysis as they ran out of com- family (Bremekamp 1954, 1966; Verdcourt puter memory when 14 600 trees were found. 1958; Robbrecht 1988, 1993) it seems that Had it been possible to save all equally Verdcourt's scheme is best supported by our parsimonious trees, their consensus could be data and our classification is in many parts much more collapsed, so it is difficult to judge similar to his. Of our 16 tribes 12 more or less to what extent the rpsl6 data alone are correspond to earlier tribal circumscriptions, phylogenetically informative. Clades not sup- two tribes have received new very different ported by bootstrap values well over 50% circumscriptions, and two are new to science. (preferably 75%) in the trees presented by The topologies of the trees are almost Andersson and Rova should not be considered identical or congruent in our four analyses. reliable. Two examples concern the circum- The resolution and support are highest in the scription and position of Spermacoceae and combined analyses, which is expected as more Theligoneae, respectively. characters are included (cf. Bremer et al. Andersson and Rova (1999) found what we 1999). In all analyses (Figs. 1--4) the same call tribe Spermacoceae paraphyletic. The seven tribes are monophyletic: Argostemma- incongruence between our studies must be of teae, Coussareeae, Ophiorrhizeae (3-data ma- the "soft incongruency" type (Seelanaen et al. trix includes only one taxon), Psychotrieae, 1997). Our tribe Spermacoceae is monophylet- Rubieae, Spermacoceae, and Urophylleae. ic and supported in most of our analyses (in Furthermore, Anthospermeae have good sup- atpB-rbcL not supported but congruent, rbcL port in the rbcL (Fig. 2) and combined an- 99%, rbcL/atpB-rbcL 99%, rbcL/atpB-rbcL/ alyses (Fig. 3) but not in the atpB-rbcL data rpsl6 100%). Andersson and Rova illustrate (Fig. 1). Lasiantheae are represented by two (in one out of 14 600 trees) the Knoxieae (in taxa in the rbcL analysis (but only one in the our data included in the Spermacoceae) as the other analyses) and are highly supported sister group to the rest of the Spermacoceae (100%; Fig. 2). Five tribes are represented by alliance. However, this relationship is not single taxa or are monogeneric and thus the supported by their data (<50%). Andersson monophyly can not be tested: Craterisper- and Rova also found Theligoneae sister to meae, Danaideae, Gaertnereae, Schradereae, Plocama, a member of the Paederieae (56%). and Theligoneae. Morindeae are monophyletic In our analyses the sister group relationship of in the combined analyses (but not supported Theligoneae is not Plocama but with the <50%; Figs. 3, 4). Finally, the tribe Pae- Rubieae, strongly supported by our data (rbcL derieae is not supported in our data, but 88%, rbcL/atpB-rbcL 96%, rbcL/atpB-rbcL/ instead paraphyletic. However, at this mo- rpsl6 97%, not supported by the atpB-rbcL). ment, we have taken a conservative approach This difference is of the "hard incongruency" and maintain Paederieae as a tribe while type (Seelanaen et al. 1997). awaiting a more detailed study (under revision The following discussion of phylogeny and by M. Backlund pers. comm.). classification is based on the tree from the Our results are also in many parts similar combined analysis of the rbcL/atpB-rbcL data to those from the recent study by Andersson (Fig. 3), unless otherwise stated, since this and Rova (1999) of rpsl6 data, but, there are analysis has the best taxon sampling relevant important differences. Notably, the results for the tribal phylogeny. 64 B. Bremer and J.-F. Manen: Rubioideae classification

The basal clades. The first dichotomy of he moved Urophylleae and Pauridiantheae to the ingroup (Fig. 3) in the tree is between a subfamily Cinchonoideae. Verdcourt was the newly circumscribed Ophiorrhizeae and the first author to include the Urophylleae in rest of the subfamily. In agreement with the Rubioideae which is in concordance with Bremer (1996a), the genus Neurocalyx (for- the present study. The Urophylleae are char- merly Argostemmateae cf. Bremer 1987) be- acterised by berries with many seeds. longs to this clade and for the first time we here Lasianthus is the next branch to split off. It show that the genera Lerchea and Xanthophy- is distinctly separated from the Urophylleae turn formerly in Pomazoteae (Bremekamp part of the tree (97%) and the rest of the 1966) or Hedyotideae (Robbrecht 1993) also subfamily (100%). The genus was earlier are closely related. The tribe is supported by a included in Psychotrieae (by most earlier high bootstrap value (92%) and is distinctly authors) or in Morindeae (Robbrecht 1988, separated (87%) from the rest of the subfam- 1993). A position within the Psychotrieae ily. One uniting morphological character for alliance has earlier been refuted by Bremer the taxa of the Ophiorrhizeae is the occurrence (1996a). As Lasianthus in our study is the sister of very small seeds (dust seeds). Andersson and to the rest of the subfamily (confirmed by an Rova (1999) found Ophiorrhiza to be sister to unpublished rbcL sequence of Lasianthus strig- Urophylleae and mention that the subfamily osus, Bremer) and unless all the remaining Urophylloideae (incl. Ophiorrhiza) "is defensi- Rubioideae taxa should be lumped into one ble, being the sister group to the rest of the tribe, Lasianthus must form a new tribe. Rubioideae, but it may not be a useful entity". Further, the genus Trichostachys belongs to We certainly agree with the latter statement this distinct branch (rbcL analysis). The but disagree with their recognition of the Lasiantheae are characterised by fleshy drupes subfamily Urophylloideae. Our data do not with 2 to 12 pyrenes, often blue or black. The support inclusion of Ophiorrhiza in the same position of Lasiantheae is supported by the 3- clade as the Urophylleae. The differences in data matrix and by the results of Andersson results might be due to taxon sampling and and Rova (1999). In their analysis the Lasian- analysis methods. We included more represen- thus branch is sister to of the tribe tatives of Ophiorrhizeae (4 genera compared to Perameae (supported by 100%). If this rela- 1) and Urophylleae (5 genera compared to 3) tionship is correct one could argue that and our analyses run to completion. Morpho- Lasianthus should be included in the tribe logically Ophiorrhizeae differ from Urophyl- Perameae instead of being elevated to a new leae by type of fruits and habit, Urophylleae tribe Lasiantheae. However, we agree with are woody with fleshy berries while Ophior- Andersson and Rova (1999) that such a taxon rhizeae never have berries and are often would be morphologically undefinable. Pera- herbaceous. ma is a genus of tiny, hairy , with very The next branch to split off (100% boot- small or reduced stipules, flowers in terminal strap value) is the tribe Urophylleae, with heads, calyx of two lobes, and dry 3-1ocular Pauridiantha nested within the tribe (as in capsular fruits with a single ovule in each Andersson and Rova 1999). Inclusion of locule. The single ovule is the only similarity to Pauridiantha within Urophylleae was first Lasiantheae, but solitary ovules occur and proposed by Verdcourt (1958) in contrast to have evolved several times in the subfamily. Bremekamp (1954, 1966) and Robbrecht The next node includes the Coussareeae (1988, 1993), who recognised Pauridiantheae (100%) as sister to the Psychotrieae alliance as a separate tribe next to the Urophylleae. (99%) and the Spermacoceae alliance (100%), These two tribes were included in the separate a relationship with low support (54%). For the subfamily Urophylloideae by Bremekamp. Coussareeae a new, highly unexpected, rela- Robbrecht did not accept this subfamily and tionship was shown between Faramea (Cous- B. Bremer and J.-F. Manen: Rubioideae classification 65 sareeae) and Coccocypselum (former Coccoc- paraphyletic. It has recently been demonstrat- ypseleae) by Bremer (1996a, Coussarea was ed by Nepokroeff et al. (1999) that Psychotria also added to this clade in Bremer and Thulin together with the other genera of the tribe 1998). Further, Nepokroeff et al. (1999) and nicely can be split into minor monophyletic Andersson and Rova (1999) showed that the groups, e.g., one includes Psychotria s.s. (sub- genus Declieuxia, formerly in Psychotrieae also genera Psychotria and Tetramerae and a belongs to this group. Another interesting Pacific group including e.g. the myrmecophil- result from the present study (and Andersson ous genera Anthorrhiza, Hydnophytum, Myrm- and Rova 1999) is the position of Cruckshank- ecodia, and Myrmephytum), another group sia (rbcL analysis) and Oreopolus (rbcL and includes the subgenus Heteropsychotria and atpB-rbcL analyses) also placed in this same the genus Palicourea. Also other genera such clade. These last two genera have been accept- as Chasallia, Chazaliella, Geophila, Rudgea, ed as a separate tribe Cruckshanksieae or they and Hymenocoleus belong to this tribe. have been included in Hedyotideae (cf. Since the relationships and circumscrip- Table 2). We have decided to include all these tions of the other tribes of the Psychotria six genera in the tribe Coussareeae despite alliance group are uncertain it is premature to morphological variation (large parts of the propose a classification. However, from our variation is found within the "Coussareeae results it is evident that the tribes Craterisper- s.s." and the "Coccocypseleae" branches, meae, Gaertnereae, Schradereae, and Morin- respectively). There are morphological traits deae all belong to this group and that they are that support their relationship, e.g., often 4- distinctly separated from Psychotrieae. Fur- merous flowers, flattened berry-like or thin- ther, all these tribes have been described walled capsular fruits, and flattened seeds. before, are accepted by several authors, and Earlier Coussareeae were supposed to have are morphologically distinct. We find it more drupaceous fruits which has been refuted (C. informative to accept them as tribes than sink Taylor pers. comm.). Interestingly all Cous- them in Psychotrieae. Craterispermeae were sareeae genera are American and most of them placed close to Psychotrieae by Verdcourt tropical. The African genus Schizocolea was (1958) but Robbrecht removed them to sub- earlier included in Coussareeae but unpub- family Antirheoideae, a position not supported lished rbcL data (Bremer) contradict that by our data. Further, from the present study as position. well as from Nepokroeff et al. (1999) and The Psyehotrieae alliance is well-support- Andersson and Rova (1999) there are no ed with a high bootstrap value (99%; Fig. 3). indications that the genus Gaertnera should This group comprises many species (probably be included in the tribe Psychotrieae as was ca 2000) which can be classified in five tribes: suggested e.g. by Verdcourt (1958, although Psychotrieae, Craterispermeae, Gaertnereae, with some hesitation) and Robbrecht (1988, Morindeae, and Schradereae. They are char- 1993). The Schradereae have recently been acterised by fleshy drupes with one ovule per expanded to include also Lecananthus, Leu- carpel (one or two in Morindeae). Many are cocodon (Puff and Buchner 1998, Puff et al. important as food sources for frugivorous 1998, Puff et al. 1998) and it seems correct that birds in the tropics (cf. Snow 1981). The they form a distinct monophyletic group. A relationships and delimitations of the Psycho- monophyletic tribe Morindeae is not contra- tria alliance are still unclear and need further dicted by our data but there is no support for investigations. The largest tribe is the Psycho- the tribe either (<50%). The delimitation of trieae s.s. which is distinctly separated from the and relationships within the Morindeae have other taxa (83% bootstrap values). Molecular been subject to recent discussions (e.g. Igers- data (Bremer 1996a) have earlier shown that heim and Robbrecht 1993). In our study we the type genus of the tribe, Psychotria, is find the Morindeae representatives in three 66 B. Bremer and J.-F. Manen: Rubioideae classification groups, corresponding to subtribe Morindinae, Bremer (1996a) that Danais belongs to the subtribe Prismatomerinae, and the Mitchella Rubioideae, and is nested within the subfamily group (Robbrecht 1993). The relationship as the sister group to the rest of the Sperma- between Mitchella and Damnacanthus of the coceae alliance separated from the other tribes Mitchella group was first pointed out by and as there are certain morphological char- Robbrecht et al. (1991) and was also highly acters that are rather rare in this part of supported in Bremer (1996a). Andersson and Rubiaceae (woody lianas, climbing shrubs or Rova (1999) included four of the tribes in the shrubs to small trees with capsular fruits Psychotrieae alliance but no representative of containing many winged seeds), we do not Craterispermeae. Their sample tree is fully hesitate to propose a new tribe. resolved. Psychotrieae and Gaertnereae are The next node in the tree is a split between shown to be well-supported, but the relation- the tribe Spermacoceae (fide Bremer 1996a) ships between the Gaertnereae, Schradereae, and the rest of the tribes. The Spermacoceae and Morindeae have only low support (59%, includes all investigated taxa of the former 55%). The monophyly of Morindeae has good tribes Hedyotideae (excluding Danais as noted support (80%), but Andersson and Rova did above), Manettieae, Knoxieae, and Sperma- not include any representative of the subtribe coceae and further also Triainolepis of the Prismatomerinae. In our 3-data analysis the former tribe Triainolepideae. None of these support for the Morindeae (including Pris- tribes can be accepted as often circumscribed matomeridae is very low (48%). (Verdcourt 1958; Bremekamp 1966; Robbrecht The Spermacoeeae alliance includes the rest 1998, 1993). Within this large group of taxa of the subfamily Rubioideae. It is a strongly there is a basal split into two highly supported supported clade (100%; Fig. 3) and represents a branches (100% bootstrap fractions for each), majority of the dry-fruited taxa of the subfam- one representing the Pentas/Triainolepis group ily. In this group we accept seven tribes and of and the other including the rest of the clade, these six (Spermacoceae, Anthospermeae, Ar- here called the Hedyotis/Spermacoce group. gostemmateae, Danaideae, Rubieae, Theligo- These two groups were initially identified by neae) are distinctly separated from each other morphological characters (Bremer 1987) and with bootstrap values ranging from 99% to supported by molecular data (Bremer et al. 100%. One tribe, the Paederieae, is found to be 1995, Bremer 1996a). The Pentas/Triainolepis biphyletic. As the tribe is under revision (M. group includes also the tribe Knoxieae (repre- Backlund pers. comm.) we refrain from doing sented by Pentanisia), a tribe Robbrecht (1988) any taxonomic changes at the moment. transferred to the subfamily Antirheoideae. At the most basal dichotomy in this Sper- Our placement of Pentanisia, and the former macoceae alliance we find the genus Danais. It Knoxieae, is corroborated by the rbcL analysis is one of the genera with winged seeds that which also includes Knoxia in the same branch. Bremekamp (1952) transferred from the One of the characters for Knoxieae was Cinchoneae to the Hedyotideae (here synonym solitary pendulous ovules, but at least ovule to Spermacoceae), a position that was accepted reduction occurs several times in this part of by Verdcourt (1958). Robbrecht (1988, 1993) the Rubioideae, also in the closely related on the other hand considered it a link between Placopoda and Carphalea. In the Hedyotis/ Rubioideae and Cinchonoideae. In a detailed Spermacoce group many of the genera have study of Danais Buchner and Puff (1993) numerous ovules and wing-less seeds, e.g., showed it to be closely related to Schismato- Hedyotis, Oldenlandia, Kohautia, and Pento- cIada and Payera. The close relationship of don, but there are also genera with many Danais and Schismatoclada has been confirmed ovules and winged seeds, earlier included in by sequence data (Bremer unpublished). The Manettieae (Bouvardia and Manettia) as well present analysis supports the conclusion from as the genera of the tribe Spermacoceae s.s. B. Bremer and J.-F. Manen: Rubioideae classification 67 with solitary, erect ovules. As shown before are related to the Rubieae and Theligoneae but (Bremer 1996a), the large genera Hedyotis and not to Anthospermeae, nor do they form a Oldenlandia are paraphyletic. The position of natural group; instead they constitute a grade Spermacoceae s.s. within the former Hedyoti- (same result in Andersson and Rova 1999). deae (pointed out by Bremer et al. 1995 and The monogeneric tribe Theligoneae is the Natati et al. 1995, Bremer 1996a) made the sister group to the Rubieae. This close Hedyotideae paraphyletic. relationship was earlier indicated by Bremer The next clade in the tree corresponds to et al. (1995) and Natali et al. (1995), although the tribe Anthospermeae, supported in the contradicted in Andersson and Rova (1999), combined analyses (100% in both analyses), however, with low support. Finally, the Rubi- the rbcL analysis (74%), and in Andersson and eae is supported by 100% in all our analyses. Rova (1999; 54%). However, in our atpB-rbcL The strong support for this group is congruent tree there is no support for the tribe. with earlier results (Natali et al. 1995, Bremer The next branch represents the new cir- 1996a, Andersson and Rova 1999). cumscription of Argostemmateae (fide Bremer Representatives of the Lathraeocarpeae 1996a), well-supported (100%), so also in remain to be investigated and the recently Andersson and Rova (1999; 99%). The former proposed position of the Perameae (Anders- circumscription of the tribe (including Neuro- son and Rova 1999) needs to be confirmed. calyx and Steenisia) was mainly based on a Material of these remaining taxa has so far supposedly unique character, adnate anthers. been difficult to obtain or analyse. With this However, that character is homoplastic and study we have nevertheless provided a com- has evolved at least three times in the family prehensive phylogeny of Rubioideae and most (in Argostemma, Neurocalyx of the Ophiorrhi- of the tribes earlier associated with the zeae, and Steenisia in the subfamily Cincho- subfamily have been investigated. A classifi- noideae, Bremer 1984). cation including ca 150 genera in 16 well- Sister to Argostemmateae is a clade con- supported monophyletic tribes summarises taining the tribes Rubieae, Theligoneae, and our results. representatives of the tribe Paederieae. The circumscription and taxonomic position of the We are grateful for all help and support; for tribe Paederieae has been much discussed, e.g., providing plant material or DNA K. Andreasen, Puff (1982) was of the opinion that the tribe is D. Bridson, M. Chase, C. Taylor, S. Malcomber, J. close to Theligoneae and Anthospermeae and McDowell, M. Kiehn, C. Puff, E. Robbrecht, C. he transferred all insect-pollinated genera from Ridsdale, O. Ryding, U. Swenson, M. Thulin, and H.-E. Wanntorp, and the directors of the botanical Anthospermeae to Paederieae. Today the tribe gardens and herbaria listed in Table 1; for technical includes ca. 15 genera (Robbrecht 1988, 1993) assistance G. Beltran, N. Heidari, and J. R6nn- but it is difficult to identify characters that holm; for useful comments on the manuscript K. unite all taxa, as most characters are variable. Bremer; for help with nomenclature problems J. We have investigated five genera molecularly Reveal and M. Thulin; latin diganoses K. Bremer; (Gaillonia, Plocarna, Putoria, Serissa, Sperrna- for financial support the Swedish Natural Science dictyon). The analyses indicate that the taxa Research Council. 68 B. Bremer and J.-F. Manen: Rubioideae classification

Appendix 1. Genera included in the Rubioideae, fide Bremer and Manen. A list of genera sorted within tribes in alphabetical order. Tribal position is indicated with the first three letters of the tribal name. Morindeae (MOR) contain also: -M, -M 1 or -P indicating subgroups. *paraphyletic taxon Genus Tribe Genus Tribe Genus Tribe Anthospermum ANT Kelloggia PAE Dibrachionostylus SPE Carpacoce ANT Plocama PAE Diodia SPE Coprosma ANT Pseudogaillonia PAE Dirichletia SPE Durringtonia ANT Pterogaillonia PAE Dolichometra SPE Eleutheranthus ANT Putoria PAE Ernodea SPE Galopina ANT Leptodermis PAE Hedyotis* SPE Leptostigma ANT Pseudopyxis PAE Hedythyrsus SPE Nenax ANT Serissa PAE Hemidiodia SPE Nertera ANT Spermadictyon PAE SPE Normandia ANT Amar acarpus PSY Hydr ophy lax SPE Opercularia ANT Anthorrhiza PSY Kadua SPE Per atan the ANT Cephaelis PSY Kno xia SPE Phyllis ANT Chasallia PSY Kohautia SPE P omax ANT Chazaliella PSY Lelya SPE Argostemma ARG Geophila PSY Lucya SPE , Mycetia ARG Hydnophyturn PSY Manettia SPE Coccocypselum COU Hymenocoleus PSY Manostachya SPE Coussarea COU Myrmecodia PSY Mitracarpus SPE Cruckshanksa COU Myrmephytum PSY Mitrasacmopsis SPE Declieuxia COU Palicouria PSY Neohymenopogon SPE Faramea COU Psychotria* PSY Nodocapaea SPE Or eopolus COU Rudg ea PSY O ldenlandia* SPE Craterispermum CRA Squamellaria PSY Otiophora SPE Danais DAN Uragoga PSY Otomeria SPE Payer a DAN Asperula* RUB Parapentas SPE Schismatoclada DAN Callipeltis RUB P aratriaina SPE Gaertnera GAE Crucianella RUB Pen tanisia SPE Pagamea GAE Cruciata RUB Pentanopsis SPE Lasiant hus LAS Didymaea RUB P entas SPE Trichostachys LAS Galium* RUB Pentodon SPE Metabolus LAS Mericarpaea RUB Phylohydrax SPE Appunia MOR-M Microphysa RUB Placopoda SPE Coelospermum MOR-M Phuopsis RUB Pseudohedyotis SPE Gynochthodes MOR-M Relbunium RUB Psyllocarpus SPE Morinda MOR-M Rubia RUB Richardia SPE Pogonolobus MOR-M Sherardia RUB Schwendera SPE Damnacanthus MOR-MI Valantia RUB Spermacoce* SPE Mitchella MOR-MI Warburgia RUB Staelia SPE Motleyia MOR-P Lecananthus SCH Synaptantha SPE Prismatomeris MOR-P Leucocodon SCH Thecorchus SPE Renellia MOR-P Schradera SCH Thyridocalyx SPE Coptophyllum OPH Agathisanthemum SPE Triainolepis SPE Ler chea OPH Amphiasma SPE Thelig onum THE Neurocalyx OPH Arcytophyllum SPE Amphidasya URO Ophiorrhiza OPH Batopedina SPE Commitheca URO Spiradiclis OPH Bouvardia SPE Maschalocorymus URO B. Bremer and J.-F. Manen: Rubioideae classification 69

Appendix 1. (Continued) Genus Tribe Genus Tribe Genus Tribe Xanthophytum OPH Carphalea SPE Pauridiantha URO Paederia PAE Chaemepentas SPE Praravinia URO Choulettia PAE Conostomium SPE Pravinaria URO Gaillonia PAE Crusea SPE Ur ophyllum URO Jaubertia PAE Den tella SPE

Appendix 2. Genera included in the Rubioideae, fide Bremer and Marten. A list of genera in alphabetical order. Tribal position is indicated with the first three letters of the tribal name. Morindeae (MOR) contain also: -M, -M1 or -P indicating subgroups. *paraphyletic taxon Genus Tribe Genus Tribe Genus Tribe Agathisanthemum SPE Hedyotis* SPE Pauridiantha URO Amaracarpus PSY Hedythyrsus SPE Payera DAN Amphiasma SPE Hemidiodia SPE Pen tanisia SPE Amphidasya URO Houstonia SPE Pentanopsis SPE Anthorrhiza PSY Hydnophytum PSY Pentas SPE Anthospermum ANT Hydrophylax SPE Pentodon SPE Appunia MOR-M Hymenocoleus PSY Peratanthe ANT Ar cytophyllum SPE Jaubertia PAE Phuopsis RUB Arg ostemma ARG Kadua SPE Phyllis ANT Asperula* RUB Ke lloggia PAE P hy lohydr ax SPE Batopedina SPE Knoxia SPE Placopoda SPE Bouv ar dia SPE Kohautia SPE P locama PAE Callipeltis RUB Lasianthus LAS Pogonolobus MOR-M Carhpalea SPE Lecananthus SCH Pomax ANT Carpacoce ANT Lelya SPE Praravinia URO Cephaelis PSY Leptodermis PAE Pravinaria URO Chaemepentas SPE Leptostigma ANT Prismatomeris MOR-P ChasaIlia PSY Lerchea OPH Pseudogaillonia PAE Chazaliella PSY Leucocodon SCH Pseudohedyotis SPE Choulettia PAE Lucya SPE Pseudopyxis PAE Coccocypselum COU Manettia SPE Psychotria* PSY Coelospermum MOR-M Manostachya SPE Psyllocarpus SPE Commitheca URO Maschalocorymus URO Pterogaillonia PAE Conostomium SPE Mericarpaea RUB Putoria PAE Coprosma ANT Metabolus LAS Relbunium RUB Cop tophyllum OPH Microphysa RUB Renellia MOR-P Coussarea COU Mitchella MOR-MI Richardia SPE Craterispemum CRA Mitr acarpus SPE Rub ia RUB Cruckshanksieae COU Mitrasacmopsis SPE Rudgea PSY Crusea SPE Morinda MOR-M Schismatoclada DAN Crucianella RUB Motleyia MOR-P Schradera SCH Cruciata RUB Mycetia ARG Schwendera SPE Damnacanthus MOR-MI Myrmecodia PSY Serissa PAE Danais DAN Myrmephytum PSY Sherardia RUB Declieuxia COU Nenax ANT Spermacoce* SPE Dentella SPE Neohymenopogon SPE Spermadictyon PAE 70 B. Bremer and J.-F. Marten: Rubioideae classification

Appendix 2. (Continued) Genus Tribe Genus Tribe Genus Tribe Dibrachionostylus SPE Nertera ANT Spiradichlis OPH Didymaea RUB Neurocalyx OPH SquamelIaria PSY Diodia SPE Nodocapaea SPE S taelia SPE Dirichletia SPE Normandia ANT Synaptantha SPE Dolichometr a SPE O ldenlandia* SPE The eor chus SPE Durringtonia ANT Opercularia ANT Theligonum THE Eleutheranthus ANT Ophiorrhiza OPH Thyridocalyx SPE Ernodea SPE Oreopolus COU Triainolepis SPE Far amea COU O tiophor a SPE Trichostachy s LAS Gaertnera GAE 0 tomeria SPE Uragoga PSY Gaillonia PAE P aederia PAE Urophyllum URO Galium* RUB Pagamea GAE Valan tia RUB Galopina ANT P alicouria PSY Warburgia RUB Geophila PSY Parapentas SPE Xanthophytum OPH Gynochthodes MOR-M Paratriaina SPE

References Bremer B. (1979) The genus Neurocalyx (Rubia- ceae-Argostemmateae) in Ceylon. Bot. Not. 132: Andersson L., Rova J. H. (1999) The rps16 intron 399-407. and the phylogeny of the Rubioideae (Rubia- Bremer B. (1984) The genus Steenisia (Rubiaceae) ceae). Plant Syst. Evol. 214: 161-186. and its taxonomic position. Nord. J. Bot. 4: 333- Andreasen K., Bremer B. (1996) Phylogeny of the 345. subfamily Ixoroideae (Rubiaceae). Opera Bot. Bremer B, (1989) The Genus Argostemma (Rubia- Belgica 7: 119-138. ceae-Argostemmateae) in Borneo. Ann. Missou- Axelius B. (1987) The genus Lerchea (Rubiaceae). ri Bot. Gard. 76: 7-49. Blumea 32: 91-114. Bremer B. (1996a) Phylogenetic studies within Ru- Axelius B. (1990) The genus Xanthophytum (Rubi- biaceae and relationships to other families based aceae) - Taxonomy, phylogeny and biogeogra- on molecular data. Opera Bot. Belgica 7: 33-50. phy. Blumea 34: 425-497. Bremer B. (1996b) Combined and separate analyses Backlund M., Oxelman B., Bremer B. (2000) of morphological and molecular data in the Phylogenetic relationships within the Gentia- plant family Rubiaceae. Cladistics 12: 21-40. nales based on ndhF and rbcL sequences, with Bremer B., Andreasen K., Olsson D. (1995) Sub- particular reference to the Loganiaceae. Am. J. familial and tribal relationships in the Rubiaceae Bot. 87: 1029-1043. based on rbcL sequence data. Ann. Missouri Bit S. S., Singh G., Gill B. S. (1984) Cromosome Bot. Gard. 82: 383-397. number reports LXXXII. In: L6ve R. (ed.) Bremer B., Jansen R. K. (1991) Comparative Rubiaceae. Taxon 33: 128-129. restriction site mapping of the chloroplast Bremekamp C. E. B. (1952) The African species of DNA implies new phylogenetic relationships Oldenlandia L. sensu Hiern et K. Schumann. within the Rubiaceae. Am. J. Bot. 78: 198- Verb. Kon. Ned. Akad. Wetensch., Afd. 213. Naturk., Tweede Sect. 48 (2): 1-298. Bremer B., Backlund M., Lantz H., Kim K. J. Bremekamp C. E. B. (1954) Les sous-familles et les (1999) More characters or more taxa for a robust tribus des Rubiac~es. Huiti6me congr~s interna- phylogeny - case study from the coffee family tional de botanique, rapports et communica- (Rubiaceae). Syst. Biol. 48: 413-435. tions, Paris Sect. 2-6: 113-114. Bremer B., Thulin M. (1998) Collapse of Isertieae, Bremekamp C. E. B. (1966) Remarks on the re-establishment of Mussaendeae, and a new position, the delimitation and the subdivison of genus of Sabiceeae (Rubiaceae); phylogenetic the Rubiaceae. Acta Bot. Neerlandica 15: 1-33. B. Bremer and J.-F. Manen: Rubioideae classification 71

relationships based on rbcL data. Plant Syst. Kirkbride J. H. (1976) A revision of the genus Evol. 211: 71-92. Declieuxia (Rubiaceae). Mem. New York Bot. Buchner R., Puff C. (1993) The genus complex Gard. 28 (4): 1-87. Danais-Schisrnatoclada-Payera (Rubiaceae). Manen J.-F., Natali A. (1996) The chloroplast Character states, generic delimitation and taxo- atpB-rbcL spacer in Rubiaceae. Opera Bot. nomic position. Adansonia 1-4: 23-74. Belgica 7: 51-57. Darwin S.P. (1976) The pacific species of Manen J. F., Natali A. (1995) Comparison of the Ophiorrhiza L. (Rubiaceae). Lyonia 1: 47-102. evolution of ribulose-1, 5-biphosphate carbox- Dwyer J. D. (1966) Notes on Coussareeae (RUN- ylase (rbcL) and atpB-rbcL noncoding spacer aceae), especially the Panamanian species. Ann. sequences in a recent plant group, the tribe Missouri Bot. Gard. 53: 368-374. Rubieae (Rubiaceae). J. Mol. Evol. 41: 920- Holmgren P. K., Holmgren N. H., Barnett L. C. 927. (eds.) (1990) Index herbariorum, Part I: The Manen J. F., Natali A., Ehrendorfer F. (1994) herbaria of the world. Regnum Veg. Phylogeny of Rubiaceae-Rubieae inferred from Igersheim A. (1992) The ovary, fruit and seed the sequence of a cpDNA intergene region. Plant development of Craterispermurn (Rubiaceae). Syst. Evol. 190: 195-211. Belgian J. Bot. 125: 101-113. Mena P. (1990) A revision of the genus Arcyto- Igersheim A., Puff C., Leint P., Erbar C. (1994) phyllum (Rubiaceae: Hedyotideae). Mere. New Gynoecial development of Gaertnera Lam. and York Bot. Gard. 60: 1-26. of presumbly allied taxa of the Psychotrieae Natali A., Manen J.-F., Kiehn M., Ehrendorfer F. (Rubiaceae): secondarily "superior" vs. inferior (1995) Phylogeny of the Rubiaceae-Rubioideae, ovaries. Bot. Jahrb. 116: 401-414. in particular the tribe Rubieae: evidence from a lgersheim A., Robbrecht E. (1993) The character non-coding chloroplast DNA sequence. Ann. states and relationships of the Prismatomerideae Missouri Bot. Gard. 82: 428-439. (Rubiaceae-Rubioideae). Opera Bot. Belgica 6: Natali A., Manen J.-F., Ehrendorfer F. (1996) 61-80. Tribal, generic and specific relationships in the Igersheim A., Rohrhofer U. (1993) The tribal Rubioideae-Rubieae (Rubiaceae) based on se- position of Otiophora (Rubiaceae): new evidence quence data of the cpDNA intergene spacer. from gynoecium structure and development. S. Opera Bot. Belgica 7: 193-203. Afr. J. Bot. 59: 431.441. Nepokroeff M., Bremer B., Sytsma K. J. (1999) Jansen S., Robbrecht E., Beeckman H., Smets E. Reorganization of the genus Psychotria (Rubia- (1996) Gaertnera and Pagarnea: genera within ceae) inferred from ITS and rbcL sequence data. the Psyhotrieae or consistituting the tribe Gae- Syst. Bot. 24: 5-27. rtnereae? A wood anatomical and palynological Olmstead R. G., Bremer B., Scott K. M., Palmer J. approach. Bot. Acta 109: 466-476. D. (1993) A parsimony analysis of the Asteridae Johansson J. T. (1987) Motleyia, a new genus of the sensu lato based on rbcL sequences. Ann. Mis- Rubiaceae from Borneo. Blumea 32: 149-155. souri Bot. Gard. 80: 700-722. Johansson J. T. (1987) Revision of the genus Puff C. (1982) The delimitation of the tribe Prismatomeris Thw. (Rubiaceae, Morindeae). Anthospemeae and its affinities to the Paederieae Opera Bot. 94: 1-62. (Rubiaceae). Biol. J. Linn. Soc. 84: 355-377. Johansson J. T. (1989) Revision of the genus Puff C. (1986) A biosystematic study of the African Rennellia Korth. (Rubiaceae-Rubioideae). Blu- and Madagascan Rubiaceae-Anthospermeae. mea 34: 3-19. Plant Syst. Evol. [Suppl.] 3: 1-535. Johansson J. T. (1994) The genus Morinda (Morin- Puff C. (1988) Observation on Carphalea Juss. deae, Rubioideae, Rubiaceae) in New Caledonia: (Rubiaceae, Hedyotideae), with particular taxonomy and phylogeny. Opera Bot. 122: 1-67. reference to the Madagascan taxa and its Kiehn M. (1986) Karyosystematic studies on Ru- taxonomic position. Bull. Jard. Bot. Belg. 58: biaceae: Chromosome counts from Sri Lanka. 271-323. Plant Syst. Evol. 154: 213-223. Puff C., Andersson L., Rohrhofer U., Igersheim A. Kiehn M. (1995) Chromosome survey of the Rubi- (1993) The tribe Schradereae (Rubiaceae) reex- aceae. Ann. Missouri Bot. Gard. 82: 398-408. amined. Bot. Jahrb. Syst. 114: 449-479. 72 B. Bremer and J.-F. Manen: Rubioideae classification

Puff C., Buchner R. (1994) Revision of Danais Snow D. W. (1981) Tropical frugivorous birds and Vent. (Rubiaceae) in Madagascar and the Co- their food plants: a word survey. Biotropica 13: mores. Adansonia 1: 11-64. 1-14. Puff C., Buchner R. (1998) Lecananthus and Swofford D. L. (1993) PAUP: Phylogenetic An- Leucocodon, two genera to be added to the tribe alysis Using Parsimony. Version 3.1. 1. Com- Schradereae (Rubiaceae). Blumea 43: 265-286. puter program. Champaign, Illinois: Illinois Puff C., Buchner R., Greimler J. (1998a) Revision Natural History Survey. of Lecananthus (Rubiaceae-Schradereae). Blu- Taylor C. M. (1996a) Taxonomic revision of mea 43: 337-346. Cruckshanksia and Oreopolus (Rubiaceae: Puff C., Greimler J., Buchner R. (1998b) Revision Hedyotideae). Ann. Missouri Bot. Gard. 83: of Schradera (Rubiaceae-Schradereae) in Male- 461-479. sia. Blumea 43: 287-335. Taylor C. M. (1996b) Overview of the Psychotrieae Puff C., Igersheim A. (1994) The character states (Rubiaceae) in the Neotropics. Opera Bot. Belg- and taxonomic position of Metabolus B1. (syn. ica 7: 261-270. Allaeophania Thw.) (Rubiaceae). Bull. Jard. Bot. Thompson J. D., Higgins D. G., Gibson T. J. Belg. 63: 241-262. (1994) CLUSTAL W: improving the sensitivity Puff C., Robbrecht E. (1989) A survey of the of progressive multiple sequence alignment Knoxieae (Rubiaceae-Antirheoideae). Bot. through sequence weighting, positions-specific Jahrb. Syst. 110: 511-558. gap penalties and weight matrix choice. Nucleic Robbrecht E. (1982) Pollen morphology of the Acids Res. 22: 4673-4680. tribes Anthospermeae and Paederieae (Rubia- Thulin M. (1997) Gaillonia (Rubiaceae-Paederieae) ceae) in relation to taxonomy. Bull. Jard. Bot. in Africa and Arabia. Nord. J. Bot. 18: 31-38. Nat. Belg. 52: 349-366. Verdcourt B. (1958) Remarks on the classification of Robbrecht E. (1988) Tropical woody Rubiaceae. the Rubiaceae. Bull. Jard. Bot. l~tat 28: 209-281. Opera Bot. Belgica 1: 1-271. Wunderlich R. (1971) Die systematische Stellung Robbrecht E., Puff C., Igersheim A. (1991) The von Theligonum. Oesterr. Bot. Z. 119: 329-394. genera Mitchella and Damnacanthus. Evidence for their close alliance; comments on the campy- lotropy in the Rubiaceae and the circumscription Addresses of the authors: Birgitta Bremer of the Morindeae. Blumea 35: 307-345. E-mail: [email protected]. Department of Schumann K. (ed.) (1891) Rubiaceae. Die natfirli- Systematic Botany, Evolutionary Biology Centre, chen Pflanzenfamilien 4 (4). (Wilhelm) Engel- Uppsala University, Norbyv. 18D, SE-752 36 mann, Leipzig. Uppsala, Sweden, Jean-Francois Manen, Seelanaen T., Schnabel A., Wendel J. F. (1997) Universit~ de Gen~ve, Conservatoire et Jardin Congruence and consensus in the cotton tribe. Botaniques, Imp6ratrice 1, CH-1292 Chamb6sy, Syst. Bot. 22: 259-290. Switzerland.